Induced pluripotent stem cells (iPSCs) are a potential source of material for cell replacement therapies. Thus, achieving maximal efficiency of reprogramming will be important for cellular medicine. In this submission we propose to test whether manipulating the mammalian glucose sensing pathway. The enzyme OGT is part of the glucose-sensing pathway and is necessary in pluripotent cells. OGT catalyzes the transfer of a sugar to target proteins, which regulates protein function. This transfer is the terminal step in the hexosamine signaling pathway (HSP). The HSP serves as a nutrient sensor, as the concentrations of the sugar donor used by OGT fluctuate with glucose levels. As a result, changes in intracellular glucose concentration cause alterations in modification of OGT target proteins. This nutrient-responsive signaling system modulates important cellular pathways, including the insulin-signaling cascade. Alterations in OGT activity are associated with diabetes mellitus and Alzheimer’s disease. Thus, our studies of role of OGT and the HSP in self-renewal and reprogramming may also shed light on the role of this pathway in other stem cells, such as neuronal and islet stem cells, whose depletion may contribute to these degenerative diseases. Understanding the role of OGT in ESCs and iPSCs will both increase our knowledge of the molecular mechanisms mammalian cells use to establish and maintain pluripotency and perhaps lead increase the efficiency of reprogramming.

Statement of Benefit to California:

Among ten leading death causes in California, five of them can directly benefit from cell-based tissue regeneration. These include heart disease, stroke, Alzheimer's disease, diabetes, and liver diseases. Currently, the economic burdens derived from these diseases are enormous. It is estimated from State of California, Department of Public Health that California taxpayers pay 48 billion dollars annually for cardiovascular diseases, 73 billion dollars excluding non-paid family care for Alzheimer's disease, and 116 billion dollars for diabetes-related diseases. Induced pluripotent stem cells (iPSCs) offer great promise as tools for regenerative medicine. However, the iPSC technology still has several shortcomings inhibiting its clinical application, one of which is the low efficiency in production iPSCs. The research outlined in this application has the potential to provide a method that substantially increases the efficiency of production of human induce iPSCs. If these studies lead to improvements in the production of iPSCs, facilitating their use in regenerative medicine, they will directly benefit the health of California citizens and reduce the economic burden presently borne by California taxpayers. This research may increase California's visibility in stem cells research and attract federal funding to sponsor future research. It may also enhance California's economic growth by stimulating the iPSC regenerative medicine industry for the treatment or cure of diseases.

Progress Report:

Year 1

Transcription factors are central for establishing the cell type-specific gene expression patterns and are often regulated by post-translational modifications (PTMs) that occur in response to developmental and environmental cues. We have found that the pluripotency transcription factor SOX2 is modified by the enzyme OGT, which is central in the glucose nutrient-sensing pathway, in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (mouse and human). Our preliminary data in the mouse system showed that mutation of the modified residue affects SOX2 function during reprogramming to pluripotency and during self-renewal of pluripotent cells. Our preliminary data also implicate this PTM in regulating human SOX2, as the equivalent residue is also modified in human pluripotent cells. We have begun experiments to gain a molecular understanding of the OGT-dependent modification, termed O-GlcNAcylation, of SOX2 in reprogramming to pluripotency in human cells. Our aims are 1) to examine whether mutation of the modified SOX2 residue enhances human reprogramming, 2) to examine whether increasing OGT amounts or activity enhance human reprogramming (since OGT is expressed at much lower levels in somatic cells than pluripotent cells), and 3) to determine the molecular consequences of expressing the mutant form of SOX2 during reprogramming and in pluripotent cells (in this third aim we use mouse as a more genetically tractable model system to first investigate molecular mechanism and then examine the mechanisms implicated in the human system). Our results to date provide a molecular basis for the increased reprogramming efficiency observed with mutant SOX2: we find that SOX2 that cannot be modified causes increased expression of epigenetic modifiers that are necessary for successful reprogramming.

Year 2

Transcription factors are central for establishing the cell type-specific gene expression patterns and are often regulated by post-translational modifications (PTMs) that occur in response to developmental and environmental cues. We have found that the pluripotency transcription factor SOX2 is modified by the enzyme OGT, which is central in the glucose nutrient-sensing pathway, in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (mouse and human). Our preliminary data in the mouse system showed that mutation of the modified residue affects SOX2 function during reprogramming to pluripotency and during self-renewal of pluripotent cells. Our preliminary data also implicate this PTM in regulating human SOX2, as the equivalent residue is also modified in human pluripotent cells. We have begun experiments to gain a molecular understanding of the OGT-dependent modification, termed O-GlcNAcylation, of SOX2 in reprogramming to pluripotency in human cells. Our aims are 1) to examine whether mutation of the modified SOX2 residue enhances human reprogramming, 2) to examine whether increasing OGT amounts or activity enhance human reprogramming (since OGT is expressed at much lower levels in somatic cells than pluripotent cells), and 3) to determine the molecular consequences of expressing the mutant form of SOX2 during reprogramming and in pluripotent cells (in this third aim we use mouse as a more genetically tractable model system to first investigate molecular mechanism and then examine the mechanisms implicated in the human system). Our results to date provide a molecular basis for the increased reprogramming efficiency observed with mutant SOX2: we find that SOX2 that cannot be modified causes i) increased expression of epigenetic modifiers that are necessary for successful reprogramming, ii) increases SOX2 association with proteins that promote pluripotency and decreases SOX2 interaction with proteins that promote differentiation, and iii) alters the genome-wide distribution of SOX2 . In addition we found that two other reprogramming factors, KLF4 and MYC, are also O-GlcNAcylated. We continue to build on these findings to understand how this nutrient sensitive modification impacts the activity of reprogramming factors to promote pluripotency.